稳定过表达miR-26a肝癌细胞株的建立

Construction of Stable Hepatocellular Carcinoma (HCC) Cell Lines Expressing miR-26a and ZsGreen Transgenes

目的建立稳定过表达miR-26a的肝癌细胞株。方法以CTE049质粒为模板,PCR扩增miR-26a序列418bp,片段两侧添加XbaⅠ和BamHⅠ酶切位点,In-Fusion克隆至pHAGE-fullEF1a-MCS-IZsGreen中,次日挑选单菌落,提取质粒并进行测序和酶切鉴定;所构建的载体命名为pLVT-miR26a。获得pLVT-miR26a后,按Invitrogen公司推荐的标准程序进行慢病毒包装和确认慢病毒是否成功生产;携带miR-26a和ZsGreen基因的慢病毒感染小鼠肝癌细胞(Hep1-6)、人肝癌细胞(BEL-7402、HepG2、Sk-Hep-1、HepG2-luc),以建立稳定过表达miR-26a的肝癌细胞系;qRT-PCR检测所建立肝癌细胞系中miR26a的表达水平。结果测序和酶切证实成功构建了pLVT-miR26a,按标准程序生产的携带miR-26a和ZsGreen基因的慢病毒上清成功感染小鼠及人肝癌细胞。结论成功建立稳定过表达miR-26a的肝癌细胞株,为相关后续研究打下了良好基础。

Objectlve To generate lentiviral expression vector harboring human miR-26a and ZsGreen genes. Methods Human miR-26a gene was amplified by PCR from the template of CTE049-21 plasmid, and subsequently cloned into the plasmid of pHAGE-fullEFla-MCS-IzsGreen to obtain the final vector of pEFla-miR26a-IRES-ZsGreen （named pLVT- miR26a） which was used to produce lentiviruses carrying miR26a and ZsGreen genes. Lentivirus harboring miR26a and ZsGreen genes was employed to infect mouse and human hepatocellular carcinoma （HCC） cell lines （i.e., Hepl-6 BEL- 7402, HepG2, Sk-Hep-1, HepG2-1uc）, followed by both GFP assay under fluorescent stereo microscope several days after infection for ZsGreen transgene expression and qRT-PCR for miR26a transgene expression. Results The lentivirus vector of pLVT-miR26a was successfully constructed. Mouse and human hepatocellular carcinoma （HCC） cell lines （i.e., Hepl-6 BEL-7402, HepG2, Sk-Hep-l, HepG2-1uc） harboring miR-26a and ZsGreen transgenes were generated showing the expression of both miR-26a and ZsGreen transgenes. Conclusion Mouse and human hepatoeeUular carcinoma （HCC） cell lines （i.e., Hepl-6 BEL-7402, HepG2, Sk-Hep-1, HepG2-1ue） stably expressing miR-26a and ZsGreen transgenes were successfully generated.